Thymic Origin of Intestinal αß T Cells Revealed by Fate Mapping of RORγt+ Cells

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Science  09 Jul 2004:
Vol. 305, Issue 5681, pp. 248-251
DOI: 10.1126/science.1096472


Intestinal intraepithelial T lymphocytes (IELs) are likely to play a key role in host mucosal immunity and, unlike other T cells, have been proposed to differentiate from local precursors rather than from thymocytes. We show here that IELs expressingthe αβ T cell receptor are derived from precursors that express RORγt, an orphan nuclear hormone receptor detected only in immature CD4+CD8+ thymocytes, fetal lymphoid tissue–inducer (LTi) cells, and LTi-like cells in cryptopatches within the adult intestinal lamina propria. Using cell fate mapping, we found that all intestinal αβ T cells are progeny of CD4+CD8+ thymocytes, indicatingthat the adult intestine is not a significant site for αβ T cell development. Our results suggest that intestinal RORγt+ cells are local organizers of mucosal lymphoid tissue.

The gut-associated immune system includes mesenteric lymph nodes (mLNs), Peyer's patches (PPs), and T lymphocytes that reside in the intestinal lamina propria (LPLs) and within the single layer of intestinal epithelial cells (intraepithelial T lymphocytes, IELs) (1, 2). T cells present in mLNs and PPs share the characteristics of mainstream peripheral αβ T cells (bearing the αβ T cell antigen receptor, TCR), whereas IELs, and to a lesser extent LPLs, are enriched in T cells that express the γδ TCR. A large proportion of αβ and γδ IELs express the CD8αα homodimeric form of the CD8 coreceptor (1, 2). In the absence of a thymus, these cells nevertheless develop and can be derived from grafts of bone marrow, fetal liver, and intestinal tissue into lymphopenic mice (35). These observations have been used to support the existence of an extrathymic pathway for the generation of IELs. However, because athymic mice have a 50 to 80% decrease in γδ IELs and an even greater reduction in αβ IELs, it has also been argued that IELs are predominantly derived from thymocytes (1, 6, 7). This view is supported by the finding that, in TCR transgenic mice, expression of self-antigen in the thymus results in deletion of conventional T cells, but selection of CD8αα+ TCR αβ IELs bearing the same antigen specificity (8).

Small clusters of hematopoietic cells, termed cryptopatches (CPs), are detected between crypts in the small intestinal lamina propria of mice and appear after weaning, numbering up to 1700 in the adult intestine (9). The majority of CP cells express c-kit and IL-7Rα, but no lineage markers (CD3B220CD11b CD11cGr-1), and have been reported to give rise to both αβ and γδ IELs when transferred to lymphopenic hosts (5, 10). Hence, it has been suggested that CP cells represent extrathymic progenitors of intestinal T cells (5, 10), although this interpretation has remained somewhat controversial (6).

The nuclear retinoic acid–related orphan receptor RORγt is necessary for the development of LNs and PPs (11, 12). During fetal life, RORγt is exclusively expressed in lymphoid tissue–inducer (LTi) cells and is required for their generation (12). In the adult, RORγt regulates the survival of immature double-positive (DP, CD4+CD8+) thymocytes (11). Using mice that are heterozygous for insertion of a green fluorescent protein (GFP) reporter into the Rorc(γt) gene [Rorc(γt)+/GFP mice] (12, 13), we observed that, in adult animals, RORγt is also expressed in cells within cryptopatches (Fig. 1A). RORγt+ cells were further detected in isolated lymphoid follicles (ILFs) (14) and in the subepithelial dome of PPs but were not evident within the intestinal epithelium, in mLNs or in periaortic LNs. Most, if not all, intestinal RORγt+ cells expressed both c-kit and IL-7Rα and linc-kit+IL-7Rα+ cells were likewise positive for RORγt (Fig. 1, B and C).

Fig. 1.

RORγt expression in the adult mouse. (A) RORγt+ cells in intestinal lymphoid tissues. Longitudinal sections of small intestine and colon of adult Rorc(γt)+/GFP mice were stained as indicated, as well as for GFP (green). CPs, small ILFs, and PPs are from the small intestine, and large ILFs are from the colon. The relative sizes of these different structures are compared in the first row. Magnifications are 400×, except for the first row and the last panel of the last row (magnification, 40×). Sections shown are representative of at least 10 individual sections and five independent experiments. (B) RORγt expression by DP thymocytes, spleen αβ T cells, and intestinal lymphoid cells. Cells from Rorc(γt)+/GFP adult mice (blue histograms) and control Rorc(γt)+/+ mice (red lines) were analyzed by flow cytometry for expression of GFP. Cells were gated as indicated. Linc-kit+IL-7Rα+ cells represented ∼0.5% of total intestinal mononuclear cells (and 0.1 to 0.2% of total PP cells). The data shown are representative of at least 10 individual mice. (C) Expression of c-kit and IL-7Rα by intestinal lin RORγt+ cells. Cells from Rorc(γt)+/GFP adult mice were analyzed by flow cytometry and gated on lin cells. Numbers indicate the percentage of cells present in each quadrant. The data shown are representative of at least 10 individual mice.

In mice rendered deficient for RORγt through breeding the Rorc(γt)GFP allele to homozygosity, intestinal linc-kit+IL-7Rα+ cells and CPs were absent, and no intestinal GFP+ cells could be observed. In these animals, ILFs also failed to develop (Fig. 2), which was apparent from the absence of B cell clusters characteristic of these structures (Fig. 1A) (9, 10). Although intestinal B cells, γδ T cells, and CD11c+ cells (Fig. 2) were present in normal numbers in the mutant mice, there was substantial and specific reduction in all subsets of intestinal αβ T cells, including CD48 (DN), CD4+, CD8αβ+, and CD8αα+ cells (Fig. 2B). This decrease in intestinal αβ T cells could be accounted for either by reduced thymic output (11) or by impaired differentiation of cells outside of the thymus. In the absence of RORγt, DP thymocytes progress prematurely into the cell cycle and undergo massive apoptosis (11), a phenotype that can be rescued by transgenic expression of Bcl-xL (11). To force expression of Bcl-xL in intestinal RORγt+ cells, we generated bacterial artificial chromosome (BAC)-transgenic mice (15) that express Bcl-xL under control of the Rorc(γt) gene [Rorc(γt)Bcl-xlTG mice] (16). In RORγt-deficient mice, this transgene was able to restore the normal cell cycle and the survival of thymocytes (fig. S1), but it failed to restore development of intestinal linc-kit+IL-7Rα+ cells (Fig. 2B), CPs, and ILFs (17). This result suggests that the mode of action of RORγt in intestinal RORγt+ cells is independent of Bcl-xL expression. Despite the absence of CPs and ILFs, relatively normal numbers of intestinal αβ T cells, including CD8αα+ TCR+ IELs, were recovered from the intestine of RORγt-deficient Rorc(γt)Bcl-xlTG mice (Fig. 2B). These results demonstrate that intestinal RORγt+ cells (i.e., linc-kit+IL-7Rα+ CP cells) are not required for development of intestinal αβ or γδ T cells.

Fig. 2.

RORγt is required for the generation of linc-kit+IL-7Rα+ cells, CPs, and ILFs. (A) T cells and lin cells from the small intestine of RORγt-expressing [Rorc(γt)+/GFP or Rorc(γt)+/+, designated wild type (wt)] and RORγt-deficient [Rorc(γt)GFP/GFP, designated RORγto] mice were analyzed by flow cytometry. Numbers indicate the percent cells present in each quadrant. The data shown are representative of at least 10 individual mice. (B) Absolute numbers of B cells, T cell subsets, and linc-kit+IL-7Rα+ cells in the small intestine of RORγt-expressing (white bars), RORγt-deficient (black bars), and RORγt-deficient, Bcl-xL transgenic (grey bars) mice. DN/4, 8αβ, and 8αα indicate the DN (CD4CD8) and CD4+, the CD8αβ+, and the CD8αα+ subsets of αβ T cells, respectively. Fifteen Rorc(γt)+/GFP or Rorc(γt)+/+ mice, 10 Rorc(γt)GFP/GFP, and 5 Rorc(γt)GFP/GFP/Rorc(γt)Bcl-xlTG mice were analyzed by flow cytometry. In statistical analyses using Student's t test, all groups are compared to the corresponding wild-type control (white bars). *P < 10–2, **P < 10–3, ***P < 10–5. In control groups (white bars), the number of αβ T cells may be overestimated because of possible contamination from remaining PP cells. (C) Longitudinal sections of the small intestine of RORγt-deficient mice were stained as indicated, as well as for GFP (green). Even though small clusters of hematopoietic (CD45+) cells were present, the absence of CD11c+ dendritic cell and B cell clusters suggests the absence of CPs and ILFs, respectively. Magnifications are 100× (two left panels) and 200× (two right panels). Sections shown are representative of at least 10 individual sections and three independent experiments.

To determine directly which cells give rise to intestinal αβ T cells, we performed a genetic cell fate–mapping experiment. BAC transgenic mice expressing Cre recombinase under control of the Rorc(γt) gene [Rorc(γt)CreTG mice] were generated and bred to R26R reporter mice, which express GFP under control of the ubiquitously active gene Rosa26 after a LoxP-flanked STOP cassette is excised by Cre (18) (Fig. 3A). Thus, in Rorc(γt)CreTG/R26R mice, only RORγt+ cells and their progeny are capable of expressing GFP. In these animals, DP thymocytes and their CD4+ and CD8+ single-positive (SP) progeny expressed GFP, whereas DN precursors did not (Fig. 3B). In spleen, all αβ T cells expressed GFP, which mapped them as the progeny of DP thymocytes. This was in contrast to γδ T cells, B cells, natural killer (NK) cells, CD11c+ dendritic cells, and CD11b+ myeloid cells, which did not express GFP (Fig. 3B, upper panel). A similar situation was observed in the intestine (Fig. 3B, lower panel), clearly demonstrating that intestinal αβ T cells were all specifically derived from RORγt+ cells.

Fig. 3.

Cell-fate mapping of RORγt+ cells. (A) Strategy for genetic cell fate mapping. Rorc(γt)CreTG mice express Cre under control of the Rorc(γt) locus on a BAC transgene. The Cre gene was inserted into the first exon of Rorc(γt). Cd4CreTG mice express Cre under control of a short synthetic promoter consisting (from 5′ to 3′) of the murine CD4 proximal enhancer, promoter, exon 1, intron 1 containing the CD4 silencer, and part of exon 2. R26R mice express GFP under control of the Rosa26 locus only after Cre-mediated excision of a LoxP-flanked STOP cassette. The Rosa26 gene is expressed ubiquitously. (B) Cells from thymus, spleen, and small intestine of adult Rorc(γt)CreTG/R26R mice (blue histograms), from the small intestine of Cd4CreTG/R26R mice (blue histograms), and from control R26R mice (red lines) were analyzed by flow cytometry for the expression of GFP. Cells were gated as indicated (B cells were gated as B220+, and NK cells as CD3DX5+ and/or NK1.1+). The data shown are representative of 8 Rorc(γt)CreTG, 5 CD4CreTG, and 10 R26R individual mice.

In a second cell fate–mapping experiment, R26R mice were bred to transgenic mice expressing Cre under the control of murine CD4 regulatory elements (19) [Cd4CreTG mice (Fig. 3A)]. In Cd4CreTG/R26R mice, all T cells that had transited through the DP stage of thymic development, such as SP thymocytes and αβ T cells in the spleen, expressed GFP (fig. S2A). Again, intestinal αβ T cells, but not γδ T cells or B cells, expressed GFP (Fig. 3B and fig. S2A). In these mice, intestinal linc-kit+IL-7Rα+ cells did not express GFP, probably because the T cell–specific minimal CD4 enhancer/promoter is not active in these cells, even though a substantial fraction of intestinal RORγt+ cells express CD4 (fig. S2B). These results confirm that, rather than being the progeny of intestinal RORγt+ cells, intestinal αβ T cells are derived from DP thymocytes. In addition, these results shed light on the source of TCR αβ IELs that express CD8αα homodimers. These unique intestinal T cells, previously proposed to be derived from DN thymocytes on the basis of experiments performed with TCR-transgenic mice (20), are shown here to differentiate from CD4+CD8+ progenitors. A synopsis of the cell fates derived from these mapping experiments is presented in table S1.

The hypothesis that CPs harbor precursors of αβ and γδ IELs (5, 10) was first questioned by the finding that linc-kit+IL-7Rα+ CP cells express germline TCR transcripts, but no pre-Tα chain (10) or RAG-2 (6). We have shown here that, indeed, intestinal αβ and γδ T cells are not derived from intestinal RORγt+ cells, which include the linc-kit+IL-7Rα+ CP cells. Although we can conclude that intestinal αβ T cells are derived from DP thymocytes, the cell fate mapping experiments do not exclude a CP-independent extrathymic origin of γδ IELs (21), because these cells are not derived from RORγt+ cells. Finally, the earlier finding that αβ IELs are present in athymic mice does not contradict our conclusions. The presence of these IELs is accompanied by the appearance of RAG+ DP T cells in mLNs, but such cells are absent in euthymic mice (6). Extrathymic T cell development may thus be a de novo pathway in lymphopenic mice, such as athymic or neonatally thymectomized mice.

Adult intestinal RORγt+ cells share all developmental, phenotypic, and functional features with fetal RORγt+ LTi cells (table S2) (12, 22). Both cell types require RORγt and the inhibitor of bHLH transcription factors, Id2, for their development (17). Furthermore, in LTα-deficient mice, LTi cells develop but do not activate mesenchymal cells and fail to induce further LN and PP development (12). Similarly, intestinal RORγt+ cells are present in LTα-deficient mice, but fail to cluster into mature CPs (fig. S3). Together, these data suggest that intestinal RORγt+ cells are the adult equivalent of fetal LTi cells. In accordance with this hypothesis, we show that intestinal RORγt+ cells are required for the development of CPs and ILFs in the adult intestine. The relation between fetal LTi, the small CPs, and the more elaborate ILFs will be important to elucidate. Although RORγt+ cells are continuously present in the intestinal lamina propria from the fetus to adulthood (fig. S4), it is unclear if they represent LTi cells that persist postnatally. It has been reported that fetal or neonatal cells with the surface phenotype of LTi cells can develop in vitro into NK cells and antigen-presenting cells (APCs) (23, 24). This is not the case in vivo, because the progeny of RORγt+ cells do not include NK cells, macrophages, or dendritic cells (Fig. 3B and fig. S2D). Because we did not find any progeny of extrathymic RORγt+ cells in the intestine or in lymphoid organs, we propose that these cells serve as organizers of lymphoid tissues, both in fetal LN and PP development and in adult CP and ILF development.

In germ-free mice, ILFs are small and harbor a majority of CP-like linc-kit+ cells (14). Moreover, the number of ILFs is increased in dextran sulfate–induced colitis in mice (25), as well as in Crohn's disease (26) and ulcerative colitis in humans (27). We therefore propose that CPs develop into ILFs in the adult intestine following inflammatory innate immune signals transmitted to the RORγt+ cells. RORγt+ may thus be an attractive therapeutic target for inflammatory bowel diseases.

Supporting Online Material

Materials and Methods

Figs. S1 to S4

Tables S1 and S2


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